A MEK inhibitor arrests the cell cycle of human conjunctival fibroblasts and improves the outcome of glaucoma filtration surgery

Better agents are needed to improve glaucoma filtration surgery outcomes compared to current ones. The purpose of this study is to determine whether mitogen-activated protein kinase kinase (MEK) inhibitors can effectively arrest the cell cycle of human conjunctival fibroblasts (HCFs) and inhibit the formation of fibrosis and scarring following glaucoma filtration surgery. A cell counting kit‑8 assay revealed that the MEK inhibitor PD0325901 exhibited concentration-dependent growth inhibition of HCFs. Quantitative PCR, immunocytochemistry, and western blotting demonstrated decreased expression of proliferating cell nuclear antigen (PCNA) and cyclin D1 and increased expression of p27 in HCFs treated with PD0325901. Flow cytometry indicated that PD0325901 arrested the cell cycle of HCFs in the G0/1 phase. The cell-migration assay showed that HCF migration rate was significantly suppressed by PD0325901 exposure. Rabbits were divided into PD0325901-treatment and control groups, and glaucoma filtration surgery was performed. Although intraocular pressure did not differ between PD0325901-treatment and control groups, bleb height was greater in the treatment group. Histopathological evaluation revealed that fibrotic changes were significantly attenuated in the PD0325901-treatment group compared to the control group. In conclusion, the MEK inhibitor impedes HCF proliferation via cell-cycle arrest and may be beneficial for glaucoma filtration surgery by reducing bleb scarring.


Effects of PD0325901 on mRNA expression
Proliferating cell nuclear antigen (PCNA) plays a crucial role in the cell cycle, particularly in DNA replication and repair 18 .PCNA is an auxiliary protein for DNA polymerase that reaches maximal expression during the S phase of the cell cycle.Cyclin D1 plays a central role in the regulation of proliferation, linking the extracellular signaling environment to cell cycle progression 19 .It is involved in the transition from the G1 phase to the S phase of the cell cycle, promoting cell cycle progression by binding to and activating cyclin-dependent kinase (CDK) 4/6, which in turn phosphorylates and inactivates the retinoblastoma protein, allowing the cell to enter the S phase.p27 is a key regulator of the cell cycle, exerting multiple functions in cell cycle control, apoptosis, epigenetic modification, and transcriptional regulation 20 .Its inhibitory effect on the cell cycle is mediated by the inhibition of CDKs, thereby preventing the progression of the cell cycle from the G1 phase.Since the decrease in cyclin D1 and PCNA, and the increase in p27 could indicate an effect of PD0325901 on cell cycle arrest in the G1 phase, these markers were investigated in this study.
Figure 2 shows the Quantitative real-time PCR (qPCR) results.PCNA expression was significantly lower in the 1-and 10-µM PD0325901 treatment groups than in the 0.1% DMSO control (P < 0.001).Compared to controls, cyclin D1 expression was significantly lower and p27 expression was significantly higher after treatment with 10 µM PD0325901 (P = 0.026, 0.013, respectively).

Effects of PD0325901 on the expression of protein level
The immunocytochemistry results are shown in Fig. 3. Treatment with PD0325901 downregulated PCNA and cyclin D1 expression but upregulated p27 expression.Figure 4 shows the western blotting results.PCNA and cyclin D1 expression were significantly lower, and p27 expression significantly higher, in both the 1 and 10 µM PD0325901 treatment groups than in the 0.1% DMSO control (all P < 0.001).

Effects of PD0325901 on cell cycle
The percentage of HCFs in the G0/1 phase was higher, and those of the S and G2/M phases were lower, in both treatment groups versus controls (Table 1, Fig. 5, P < 0.008).

Effects of PD0325901 on migration assay
Figure 6a shows typical images of the cell-migration assay.There were no differences in scratch width between the DMSO and PD0325901 groups at up to 24 h.However, at 48 h, the scratch disappeared in all controls but remained in all PD0325901 treatment samples; the scratch widths significantly differed between the treatment groups and controls (Fig. 6b, P < 0.001).

Effects of PD0325901 on a glaucoma filtration model in rabbits
Representative photographs after glaucoma filtration surgery are shown in Fig. 7.The PD0325901 treatment group showed less hyperemia and more diffuse blebs than the control group.It also had a greater average bleb height on postoperative day 7 (Fig. 8).Although IOP tended to be lower in that group, it did not significantly differ from the control on any of the measurement dates (Fig. 9).No differences were observed between the treatment and control groups in slit-lamp and fundus examinations.Hematoxylin and eosin (HE) staining revealed densely packed cells in the bleb area of controls, suggestive of fibrosis (Fig. 10a, b).Conversely, in the treatment group, the cells were relatively sparse and scar formation was suppressed (Fig. 10c, d).Elastica van Gieson (EVG) staining revealed densely stained collagen deposits and scar formation in both the conjunctiva and sclera of controls (Fig. 10e, f).By contrast, in the PD0325901 treatment   and 2. Samples from the same experiment were used, and gels and blots were processed simultaneously.

Discussion
We investigated whether PD0325901, a MEK inhibitor, could arrest the HCF cell cycle and prevent scar formation after glaucoma filtration surgery.The CCK-8 assay revealed concentration-dependent growth inhibition of HCFs by PD0325901.Immunocytochemistry, qPCR, and western blotting showed that PD0325901 downregulated cyclin D1 and PCNA expression, and increased p27 expression, in HCFs.Flow cytometry indicated that it arrested the HCF cell cycle in the G0/1 phase, while the migration assay revealed that it inhibited migration of HCFs in vitro, implying an inhibitory effect on wound healing.In a rabbit model of glaucoma filtration, PD0325901 effectively maintained bleb volume and prevented fibrotic scarring.ERK1/2 is ubiquitously expressed and is part of the Raf/MEK/ERK signal transduction cascade 21 .This cascade plays a crucial role in cell-cycle progression, cell migration, proliferation, and transcription 21 .The in vitro experimental results of our study reveal the effects of a MEK inhibitor in terms of arresting the HCF cell cycle and inhibiting cell proliferation.
MEK and p38 are involved in the mitogen-activated protein kinase (MAPK) cascade 22 .p38 is activated by oxidative stress and inflammatory cytokines, and is involved in the regulation of cellular responses such as inflammation, apoptosis, and differentiation 22 .It has been shown that p38 inhibitors suppress myofibroblast transdifferentiation of HCFs and are effective for treating a rabbit model of glaucoma filtration surgery 23,24 .The MEK cascade differs from p38 in that it is activated by growth factors and hormones and is involved in regulating cell proliferation 22 .Wen et al. demonstrated that another MEK inhibitor, U0126, suppressed myofibroblast transdifferentiation of HCFs, as well as TGF-β1-induced phosphorylation of Smad2/3, p38/MAPK, and ERK1/2 17 .Thus, MEK inhibitors, as with p38 inhibitors, may effectively regulate postoperative fibrosis after glaucoma filtration surgery through the modulation of scar formation via the MAPK cascade.
In addition to the above-described mechanisms via signaling-pathway suppression, we found that PD0325901 attenuated fibrosis by arresting the HCF cell cycle.PD0325901 significantly blocked the cell cycle and prevented cell proliferation but did not cause HCF cell death.It did not show high cytotoxicity compared to other antifibrotic drugs, such as MMC, implying that it can be used safely in combination with other antifibrotic drugs in glaucoma surgery.We also showed the effective suppression of fibrosis in a rabbit model of glaucoma filtration surgery via subconjunctival injection of PD0325901.Regarding the relationship between cell-cycle regulation and the suppression of fibrosis after filtration surgery, a recent study also showed that subconjunctival injection of adrenaline during trabeculectomy or other filtration surgeries effectively suppressed the contractile properties of HCFs and inhibited fibroblast proliferation by blocking key cell-cycle genes 25 .MEK inhibitors have been widely used for the treatment of neoplastic disorders, with the expectation that they suppress tumor cell growth [14][15][16] , particularly in combination with other therapeutic agents.As currently used drug therapies, 5-FU and MMC The administration of multiple inhibitors has the potential for synergic effects 26 .For example, we previously reported the effects of an mTOR inhibitor on HCF proliferation and suppression of fibrosis after filtration surgery in rabbits 27 .Meanwhile, a synergistic antiproliferative effect has been reported for the combined use of an mTOR inhibitor and MEK inhibitor in tumor cells 26 .Thus, the combination of several inhibitors may be useful for the safe and effective suppression of HCF proliferation.5-FU inhibits the production of deoxythymidine monophosphate, which is essential for DNA synthesis, contributing to its cytotoxic effects 28 .MMC is an alkylating agent that crosslinks DNA, thereby directly causing DNA damage and interfering with DNA synthesis and cell division 1 .In contrast, MEK inhibitors target the MEK/ERK pathway, which is a key signaling pathway involved in cell proliferation and survival 11 .The use of DNA-damaging reagents such as MMC induces activation of ERK 29 , which has also been suggested to contribute to cell cycle re-entry after DNA damage-induced cell cycle arrest 30,31 .Hence, MEK inhibitors are expected to be useful as adjuncts to MMC.Further research is needed to explore this topic.
Although in our study IOP tended to be lower in the PD0325901 group, there were no significant differences between the treatment and control groups at any time points.In the rabbit strain used in this study, the baseline IOP was so low that any IOP-lowering effect via sustainable bleb formation with PD0325901 could not be verified.In a future study, we plan to use rabbit or other animal models of ocular hypertension to assess this effect.
This study had several limitations.First, we did not compare PD0325901 with currently used perioperative agents such as MMC or 5-FU.Second, the postoperative observation period was short.Because bleb failure in rabbits has been reported to occur within 10-14 days postoperatively without the use of antimetabolites, the observation period was set at 7 days 32 .Long-term observation is necessary for comparing the effect of PD0325901 and antimetabolites on scar formation.In addition, a sustained drug-delivery system may be useful and should be explored in the future.Third, the side effects of PD0325901 have not been adequately observed.MEK inhibitors can cause retinopathy and uveitis 33 .Vitreous injection of PD0325901 at concentrations much higher than those tested in this study causes retinopathy in rabbits 34 .Since there is a report of MEK inhibitor hindering wound healing of corneal epithelial cells 35 , corneal damage may need to be carefully monitored when using MEK inhibitor intraoperatively.Although we observed no adverse effects, additional studies are needed to determine the safe dose of PD0325901.Forth, two bands of different molecular weights are observed in the p27 membrane in the supplement figure.p27 isoform has been reported 36 , and bands at similar molecular weight positions was found in a previous report 37 .The results of this study may be due to the p27 isoform.
In summary, PD0325901 arrests the cell cycle of HCFs and may be beneficial in suppressing scar formation after glaucoma filtration surgery.

Cell culture and passage
Primary HCFs were obtained from human donor eyes and characterized as described previously 38 .The cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 containing 10% FBS and antibiotic-antimycotic solution (100×) (Sigma-Aldrich, St. Louis, MO, USA) in a CO 2 incubator at 37 °C.In all experiments, only cells from passages 3-6 were used.To arrest the cell cycle, HCFs were grown to confluence and starved for 48 h in DMEM/F-12 without FBS.

Cell proliferation assay
The cytotoxicity and growth-inhibition effects of PD0325901 were assessed using a CCK-8 assay.After cellcycle arrest, 1 × 10 4 cells were replated in a 96-well plate in DMEM/F-12 medium with 10% FBS with or without PD0325901 (100 nM-100 µM dissolved in 0.1% DMSO or 0.1% DMSO in phosphate-buffered saline (PBS) in a CO 2 incubator at 37 °C.After incubation for 24, 48, 72, or 96 h, 10 µL CCK-8 solution was added to each well and incubated at 37 °C for 2 h.The absorbance at 450 nm (OD450) was measured using a multimode plate reader (Multi Microplate Reader, ARVOX3; Parkin Elmer, MA, USA).

Quantitative PCR
Following serum starvation, 5 × 10 4 cells were replated in a 24-well plate in DMEM/F-12 medium with 10% FBS with or without PD0325901 (1 or 10 µM).After incubation for 48 h, the cells were lysed using ISOGEN (NIP-PON GENE Ltd., Tokyo, Japan), and isopropyl alcohol and chloroform were used to extract mRNA.qPCR was performed as described previously 27 .Primers were purchased from Hokkaido System Science (Hokkaido, Japan).

Western blotting
After cell-cycle arrest, 1 × 10 6 cells were replated in DMEM with 10% FBS with or without PD0325901 (1 or 10 µM) or 0.1% DMSO in a 60-mm dish, and incubated for 48 h.The cells were obtained using radioimmunoprecipitation assay buffer (RIPA buffer; Thermo Fisher Scientific K.K., Kanagawa, Japan) and protease inhibitors (Roche Diagnostics, Basel, Switzerland).A BCA Protein Assay Kit (Thermo Fisher Scientific K.K.) was used to ascertain the protein quantities in the supernatant.Western blotting was performed as described previously 27 .

Cell cycle analysis
After serum starvation, 3 × 10 6 cells were replated in DMEM with 10% FBS with or without PD0325901 (1 or 10 µM) or 0.1% DMSO in a 100-mm dish, and then incubated for 48 h.The cells were trypsinized, washed in PBS, and fixed with 70% ethanol at -20 ℃.Fixed cells were stained with 50 mg/mL propidium iodide.Flow cytometric analysis was performed using a BD FACSAria™ III cell sorter (BD Biosciences, San Jose, CA, USA).The percentage of HCF in each cell cycle was obtained by the Watson Pragmatic algorithm in FlowJo v10.

Migration assay
Confluent HCFs in a 24-well plate were serum starved for 48 h.Each well was scratched crosswise with a pipette tip.After washing with PBS, the medium was changed to DMEM/F-12 with PD0325901 (1 or 10 µM) or 0.1% DMSO.Scratches were photographed at 0, 24, and 48 h after stimulation using a microscope (BZ-9000; Keyence

Figure 1 .
Figure 1.Effects of PD0325901 on toxicity and proliferation.(a) To assess toxicity, HCFs were incubated for 24 h with PD0325901 (100 nM-100 µM or 0.1% DMSO, after which the OD450 was measured using the CCK-8 assay.No significant differences were observed among the groups.(b) To evaluate proliferation, HCFs were incubated for up to 96 h with PD0325901 or 0.1% DMSO, after which the OD450 was measured using the CCK-8 assay.The points in this figure show the average OD450 at each PD0325901 concentration or 0.1% DMSO.PD0325901 inhibited HCF proliferation in a concentration-dependent manner.

Figure 3 .Figure 4 .
Figure 3. Effects of PD0325901 on immunocytochemistry.Expression of PCNA and cyclin D1 was lower, and p27 expression was higher, in PD0325901-treated HCFs compared to DMSO-treated HCFs.

Figure 5 .
Figure 5. Effects of PD0325901 on cell cycle.Treatment with both 1 and 10 μM PD035901 increased the percentage of HCFs in the G0/1 phase and decreased the percentages of HCFs in the S and G2/M phases compared to the 0.1% DMSO control (all P < 0.008).

Figure 6 .Figure 7 .
Figure 6.Effects of PD0325901 on migration assay.The cell-migration assay was performed on HCFs for 0, 24, and 48 h (n = 6).The baseline (0 h) represented the migration distance of cells with no stimulation.(a) The scratch width did not differ among the groups at 24 h.(b) At 48 h, all DMSO-group replicates showed reduced widths whereas all treatment replicates retained their widths; migration was significantly suppressed by 1 and 10 μM PD0325901 exposure at 48 h post-incubation compared to the DMSO control (P < 0.001).

Figure 8 .Figure 9 .
Figure 8. Anterior segment optical coherence tomography (AS-OCT) after glaucoma filtration surgery.(a)The space in the bleb in the PD0325901 treatment group was better maintained compared to that in the DMSO control group.(b) Bleb heights on postoperative day 7 were significantly greater in the treatment group than in controls (P = 0.003).

Figure 10 .
Figure 10.Histopathologic evaluation of blebs.HE staining of eye tissue revealed dense cells and fibrotic scarring in the control group (a,b) and relatively sparse cells and milder scar formation in the treatment group(c,d).EVG staining revealed dense collagen deposition and scar formation in the conjunctiva and sclera of the control group (e,f), whereas collagen deposition was faintly stained, conjunctival tissue was loose, and the subconjunctival space was maintained in the treatment group (g,h).Magnification: ×4 for (a,c,e,g); ×10 for (b,d,f,h).Arrows: the conjunctiva, arrow heads: the sclera.